The ability of the hematopoietic stem cell to function as a source of committed progenitors throughout the lifetime of the organism is, at present, a poorly understood phenomenon. The major characteristic of the hematopoietic stem cell is its ability to self renew in the absence of differentiation (Morrison et al., Ann. Rev. Cell Dev. Biol., 11, 35-71 [1995]). This self renewal phenomenon is especially remarkable in light of the fact that the hematopoietic stroma, which is in close physical contact with the stem cell, is known to be a source that is rich in factors which mediate the growth and differentiation of hematopoietic progenitors (Deryugina and Muller-Sieberg, Crit. Rev. in Immunol. 13(2), 115-150 [1993]). For example, a recent PCR analysis of hematopoietically active endothelial cell stromal lines derived from the murine yolk sac revealed that these cells produced a plethora of growth and differentiation factors including stem cell factor, FLT 3 ligand, M-CSF, LIF and IL-6 (Fennie et al., Blood 86(12), 4454-4467 [1995]). Such growth factors, in addition to many others, are known to induce the expansion and differentiation of stem cells, and these endothelial cell lines induced a rapid expansion and differentiation of embryonic hematopoietic stem cells along the myeloid pathway, although very early progenitor cells are also amplified by these stromal cells (C. Fennie and L. Lasky--unpublished data). It has also been shown that incubation of highly purified stem cell populations in the presence of various purified hematopoietic growth factors induces differentiation with subsequent loss of the cells' ability to competitively repopulate the hematopoietic compartment of lethally irradiated animals, consistent with the induction of terminal differentiation (Peters et al., Blood 87(1): 30-37 [1996]). Thus, the stem cell, whether in an embryonic or adult stromal environment, must maintain an undifferentiated state in spite of the fact that it is being exposed to a variety such maturation factors (Deryugina and Muller-Sieberg, supra).
Although the hematopoietic growth factors are very diverse both structurally and functionally, they are all believed to play a role in mediating protein phosphorylation (Paulson and Bernstein, Semin Immunol. 7(4), 267-77 [1995]). This protein modification can occur via direct means, such as in the cases of the stem cell factor and FLT-3 receptors, both of which have intrinsic tyrosine kinase activity, or via indirect means, as is the case of the hematopoietic/cytokine growth factor receptors for, for example, IL-3, EPO and TPO. In the case of the hematopoietic/cytokine growth factor receptors, tyrosine phosphorylation is indirectly accomplished by the activation of the JAK kinases, which occurs after growth factor mediated receptor dimerization (Ihle et al., Annu. Rev. Immunol. 13, 369-398 [1995]). In both cases, diverse complex pathways of protein phosphorylation are stimulated upon receptor binding. The intrinsic tyrosine kinase receptors mediate their signals via an elaborate series of tyrosine phosphorylation events which ultimately activate the RAS signaling pathway (Fantl et al., Ann. Rev. Biochem. 62, 453-481 [1993]). This pathway eventually leads to the activation of the serine/threonine specific MAP kinase pathway which results in transcriptional activation. In contrast to this intricate pathway, hematopoietic growth factor-induced receptor dimerization mediates more direct activation events. Thus, the stimulation of the JAK kinases by receptor binding leads to the tyrosine phosphorylation and subsequent dimerization of various STAT proteins. These activated STAT proteins than migrate to the nucleus, bind to STAT responsive sites in the nuclear DNA and induce transcription of differentiation and growth specific genes. Thus, a major effect of the growth factors produced by the hematopoietic stroma is to mediate the activation of various cellular pathways by protein phosphorylation.
The regulation of protein tyrosine phosphorylation is accomplished by a balance between protein tyrosine kinases and protein tyrosine phosphatases (PTPs) (Walton and Dixon, Ann. Rev. Biochem. 62, 101-120 [1993]; Sun and Tonks, Trends Biochem. Sci., 19(11), 480-485 [1994]). All PTPs contain a phosphatase domain including a subset of highly conserved amino acids, and a recent crystal structure analysis of PTP 1B complexed with a tyrosine phosphorylated peptide revealed that many of these conserved residues are involved with substrate recognition and tyrosine dephosphorylation (Jia et al., Science 268(5218), 1754-1758 [1995]). PTPs fall into two general categories: receptor type and non-receptor type. The receptor type PTPs have variously sized extracellular domains and, generally, two intracellular phosphatase domains Walton and Doxin, supra; Sun and Tonks, supra. The extracellular domains often contain a number of motifs that are generally utilized in cell adhesion including immunoglobulin domains and fibronectin-like regions. Many of these PTPs appear to function as homotypic and heterotypic sensors of the extracellular space, and they have been hypothesized to play roles in contact inhibition, cell guidance and other intercellular functions (Brady-Kalnay and Tonks, Curr. Opin. Cell. Biol. 7(5), 650-657 [1995]). The non-receptor PTPs are generally intracellular enzymes. They have various cellular localizations, depending upon the types of domains they contain, and some of the enzymes contain SH2 motifs which allow them to interact intimately with phosphotyrosine residues. While many of the non-receptor PTPs are in various cytoplasmic locations, a small number of these enzymes are found in the nucleus Flores et al., Mol. Cell. Biol. 14(7), 4938-46 [1994]). Many non-receptor PTPs appear to function as both activators as well as inhibitors of diverse tyrosine phosphorylated proteins. A subset appear to play important roles in hematopoiesis. For example, the motheaten mouse, which has a phenotype of lethal myeloid amplification and inflammation, has been found to have a mutation in the PTP 1C gene Shultz et al., Cell 73(7), 1445-54 [1993]; McCulloch and Siminovitch, Adv. Exp. Med. Biol. 365, 245-54 [1994]). In addition, the level of tyrosine phosphorylation of the EPO receptor, as well as the level of receptor activation, appears to be in part controlled by the PTP 1C enzyme as well Klingmuller et al., Cell 80(5), 729-38 [1995]). However, while these examples, as well as others, highlight the potential importance of the PTPs, very little is known regarding the physiological importance of these enzymes.